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Single-molecule force spectroscopy often refers to the measurement of the enzymatic activities coupled to the mechanical properties of biomolecules. We apply a simple dark-field microscope to a single-molecule flow-stretching method that quantitatively measures thermal fluctuations of the micron bead linked to DNA under a hydrodynamic force. Using various lengths of DNA attached to the bead, we measured the external force exerted on DNA immobilized on the surface and quantitatively showed the anticorrelation between the thermal fluctuation and the length of DNA. We propose a multiplexed single-molecule flow-stretching technique with better spatial resolution.

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.

Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM., Cas12a is a RNA-guided DNA endonuclease whose detailed mechanisms of target searching and DNA cleavage remained unclear. Here authors use single-molecule fluorescence assays to show that Cas12a searches for their on-target site.

Summary The αɛθ core of Escherichia coli DNA polymerase III (Pol III) associates with the β 2 sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The α subunit is the polymerase while ɛ is the 3′ to 5′ proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood. We used single-molecule assays to determine the excision rate and processivity of the β 2 -associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ɛ and β 2 . Thus, the ɛ-β 2 contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between ɛ and β 2 .

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that Taq-MutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.

Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3′- or 5′-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5′-MMR excision reaction requires the HsMSH2–HsMSH6 heterodimer, the 5′ → 3′ exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1–HsPMS2 heterodimer substantially influences 5′-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2–HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1–HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5′ MMR.

We have investigated the progress of structural distortions in DNA molecules by single-shot coherent diffraction using extreme-ultraviolet radiation from a free-electron laser. A speckle pattern of DNA molecules was successfully acquired using photons in a single pulse with a 100 fs pulse width. The radiation damage was assessed by a cross correlation, revealing that the first exposure has significantly deformed most of the original structures. Molecules were not completely destroyed by the first single-shot exposure and underwent subsequent distortions through continued exposure, until eventually deforming into a radiation-hard structure.

MutL stimulates the DNA duplex unwinding activity of UvrD in methyl-directed DNA mismatch repair (MMR) via their physical interactions. However, the molecular functions of MutL associated with the DNA binding and UvrD helicase have been partially understood. We present the kinetic characteristics of the single-stranded DNA (ssDNA) binding activity of MutL in the absence or the presence of UvrD helicases using the single-molecule techniques. The lengthening of the ssDNA due to the ssDNA binding of MutL allows us to observe association and dissociation of MutL from the ssDNA in real-time. In this study, we demonstrate that the nonspecific ssDNA binding of MutL can be involved in subsequent loading of UvrD helicases onto the ssDNA in a manner independent of ATP hydrolysis of MutL.